COMPARATIVE ANALYSIS OF HISTOPHILUS SOMNI IbpA WITH OTHER FIC ENZYMES REVEALS DIFFERENCES IN SUBSTRATE AND NUCLEOTIDE SPECIFICITIES

A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine mono-phosphate (AMP) to Rho GTPases (Yarbrough et al., 2009, Science, 323:269; Worby et al., 2009, Mol. Cell, 34:93). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the protein IbpA from the pathogenic bacterium Histophilus somni adenylylates mammalian RhoGTPases, RhoA, Rac1 and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of IbpA’s Fic2 domain and compare its activity to other known Fic adenylyltransferases, VopS from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE. We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage, and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.